伯氏疏螺旋体PD91外膜蛋白C克隆表达及序列分析

目的 构建伯氏疏螺旋体PD91菌株外膜蛋白C(OspC)的表达载体，克隆表达OspC，用于莱姆病的预防、诊断和致病机理上的研究。方法用PCR扩增PD91 ospC基因，定向克隆到表达载体PET-11D，构建重组质粒。采用酶切分析及序列测定等方法鉴定重组质粒的正确性。结果 ospC基因被正确克隆到表达载体PET-11D中。序列测定结果证实与已报道的ospC基因序列同源性介于62％～86％之间。结论我国PD91菌株的ospC编码基因与已报道菌株的ospC菌株在同源性上存在较大的差异。PET-11D—ospC重组质粒的成功构建为我国莱姆病的进一步研究莫定了基础。     

中国莱姆病螺旋体伽氏疏螺旋体PD91鞭毛蛋白的基因克隆和表达

目的 对中国莱姆病螺旋体伽氏疏螺旋体基因种参照菌株PD91的鞭毛蛋白编码基因进行克隆表达与基因序列分析。方法 设计引物,用聚合酶链反应(PCR)技术获得PD91的鞭毛蛋白编码基因片段,经酶切、连接,插入质粒pET-11d中,构成重组质粒pET11d-fla,转化致大肠埃希菌BL21,提取质粒进行酶切、DNA测序和氨基酸序列分析鉴定,诱导表达,筛选高效表达株,应用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(WB)方法,鉴定重组蛋白及其抗原性。结果成功地获得了鞭毛蛋白的编码基因片段和基因重组,重组蛋白在宿主菌BL21中高效表达。WB结果显示重组鞭毛蛋白与PD91的鞭毛蛋白具有相同的抗原性。经测序显示该鞭毛蛋白的基因片段长度为1011bp,与北美莱姆病螺旋体标准株B31的DNA碱基序列及氨基酸序列比较分析,同源性分别为94.70％、95.85％。结论 在国内首次成功地对中国莱姆病螺旋体伽氏疏螺旋体基因种的鞭毛蛋白基因进行了克隆表达,并证实具有良好的抗原性,为莱姆病血清学诊断研究提供了较好的基础。     

中国莱姆病螺旋体PD91重组OspC的鉴定和抗原性检测

目的 重组中国莱姆病螺旋体PD91外膜蛋白C(OspC)并在大肠埃希菌中表达，用于早期莱姆病诊断的研究。方法 用聚合酶链反应(PCR)扩增出PD91外膜蛋白C基因，定向克隆到表达载体PET-llD，构建重组质粒PET-llD-ospC。用PCR、限制性内切酶分析及序列测定等方法鉴定重组质粒。用Western Blot检测其抗原性。结果 OspC基因被正确克隆到表达载体PET-llD中。序列测定结果证实与国外已报道的序列存在一定的差异。OspC具有强抗原性。结论 该项研究为国内莱姆病的早期特异性诊断的研究奠定了基础。     

从桂黔交界地区中华硬蜱中分离到莱姆病螺旋体

目的从桂黔交界地区捕获的蜱中分离莱姆病螺旋体,以探查广西壮族自治区（广西区）和贵州省莱姆病的传播媒介。方法用BSK-Ⅱ培养基从蜱中分离莱姆病螺旋体。用PCR扩增分离菌株的5S～23SrRNA基因间隔区,将PCR扩增产物克隆后测序,通过将测序结果与基因库中莱姆病螺旋体菌株的5S～23SrRNA基因间隔区进行同源性比较和分析,从而鉴定分离株的基因型。结果从捕获的中华硬蜱中分离出1株莱姆病螺旋体（命名为QLT1）。分离株经鉴定为Borrelia valaisiana基因型莱姆病螺旋体。结论中华硬蜱很有可能是广西区和贵州省莱姆病的传播媒介。     

重组伯氏疏螺旋体鞭毛抗原用于莱姆病血清学诊断方法的研究

以重组伯氏疏螺旋体鞭毛抗原的特异性区段为诊断抗原,选取从疫区采集的３０份阳性血清实验标本,并选择相关的对照,建立起间接法ＥＬＩＳＡ和双抗夹心ＥＬＩＳＡ。结果显示,两种方法匀显示出了高特异性,而双抗夹心ＥＬＩＳＡ敏感性达６０％,优于间接法ＥＬＩＳＡ。证明该“平截性蛋白”作为诊断抗原的优越性及其应用的可行性,尤其是作为早期莱姆病的筛检方法。     

Risk of Lyme disease: perceptions of residents of a Lone Star tick-infested community.

BACKGROUND: Lone Star ticks (Amblyomma americanum) have been suggested as a vector of the agent of Lyme disease (Borrelia burgdorferi sensu lato) in the USA, based on associations with an infection manifesting mainly as erythema migrans. In laboratory experiments, however, they failed to transmit B. burgdorferi sensu stricto. METHODS: In this study, carried out from 1994 to 1996, we determined the seroprevalences of B. burgdorferi (1.2%), Ehrlichia chaffeensis (7%), E. phagocytophila (0%), Rickettsia rickettsii (0%), R. typhi (0%), Coxiella burneti (0%), Francisella tularensis (0%), and Babesia microti (0%) by standard serological methods for 325 residents (97% of the total population) of Gibson Island, coastal Maryland, USA, where 15% of the residents reported having had Lyme disease within a recent 5-year span. FINDINGS: Of the 167 seronegative individuals who were followed up prospectively for 235 person-years of observation, only 2 (0.85%) seroconverted for B. burgdorferi. Of 1556 ticks submitted from residents, 95% were identified as Lone Star ticks; only 3% were deer ticks (Ixodes dammini), the main American vector of Lyme disease. B. burgdorferi s.s. infected 20% of host-seeking immature deer ticks, and borreliae ("B. lonestari") were detected in 1-2% of Lone Star ticks. Erythema migrans was noted in 65% of self-reports of Lyme disease, but many such reports indicated that the rash was present while the tick was still attached, suggesting a reaction to the bite itself rather than true Lyme disease. Sera from individuals reporting Lyme disease generally failed to react to B. burgdorferi or any other pathogen antigens. CONCLUSION: The residents of Gibson Island had an exaggerated perception of the risk of Lyme disease because they were intensely infested with an aggressively human-biting and irritating nonvector tick. In addition, a Lyme disease mimic of undescribed etiology (named Masters' disease) seems to be associated with Lone Star ticks, and may confound Lyme disease surveillance. The epidemiological and entomological approach used in this study might fruitfully be applied wherever newly emergent tickborne zoonoses have been discovered.     

北京奥运会期间的一例输入性莱姆病病例分析

目的分析拟诊莱姆病患者血清学检查结果,为有效诊治莱姆病提供科学依据。方法应用间接免疫荧光试验（IFA）、ELISA和蛋白免疫印迹（WB）3种实验方法,对此例患者进行血清抗伯氏疏螺旋体IgM和IgG抗体检查,并结合临床疾患进行综合分析。结果 3种试验方法IgG均为阳性,其中IFA的IgG抗体滴度达到1：128,WB的IgG有2条蛋白带P83和P39;3种试验方法IgM均为阴性。结论患者临床发展病程符合莱姆病的特征,实验室结果阳性,莱姆病的诊断可以成立。     

泉州市莱姆病的发现及蜱媒与宿主的调查研究

[目的]凋查泉州市莱姆病的病原、传播媒介、宿主动物及人群感染情况。[方法]以流行病学、血清学、病原学等疗法调查泉州市部分地区人群感染以及鼠类、野兔及牛等动物体表的蜱类,用PCR法检测其伯氏疏螺旋体DNA。[结果]首次确认泉州市存在莱姆病病例,安溪、永春等县林区人群存在感染,从粒形硬蜱中检出伯氏疏螺旋体DNA;从黄毛鼠、白腹巨鼠及野兔查出特异性抗体。[结论]从流行病学和病原学上证实泉州市存在莱姆病及其媒介与宿主,是当地一种新发现的人兽共患传染病。     

Identification and characterization of Borrelia antigens as potential vaccine candidates against Lyme borreliosis

The three Borrelia species, Borrelia afzelii, Borrelia burgdorferi and Borrelia garinii are the main species causing the most common tick-borne zoonosis, Lyme borreliosis. By applying a genomic approach relying on human antibodies we have identified 122 antigenic Borrelia proteins associated with Lyme borreliosis, including already known and published protective antigens. The heterogeneity of the Borrelia species causing Lyme borreliosis makes the search for conserved antigens providing broad protection challenging. Using several in vitro assays we narrowed down the selection to 15 vaccine candidates. These antigens were further analyzed for antigenicity and cross-reactivity using sera from mice infected with the three pathogenic Borrelia species. All antigens analyzed showed a high degree of cross-reactivity between the three Borrelia species, essential for providing cross-protection. We also investigated whether mice infected with B. afzelii through tick exposure are primed to mount cytokine responses. For a selection of these antigens, we observed preferentially a pro-inflammatory response in C3H/HeN mice, while in contrast also a type 2 T cell response was seen in the Borrelia-resistant mouse strain BALB/c. Thus, antigens mounting a type 2 or mixed type 2/type 1 T cell response might be preferred vaccine candidates for evaluation in animal models of Lyme borreliosis.     

湖南省平江县莱姆病调查研究

目的：调查湖南省平江县莱姆病的分布情况。方法：采用间接免疫荧光抗体法和直接荧光抗体染色法,于1999－2000年在平江县选点调查。结果：共查山林地区居民500人,血清抗莱姆病螺旋体抗体（IgG)阳性者32人,阳性率6.40%。38例临床和血清学确诊的莱姆病患者,其主要的临床表现有环形或慢性游走性红斑、关节炎、面神经麻痹、多发性神经炎、脑膜炎和心脏损害等。当地的蜱类以二棘血蜱为优势种,占85％,该蜱中肠携带莱姆病螺旋体率为12.0%(6/50)。结论：平江县存在莱姆病的自然疫源地,人群中有莱姆病的发生和流行。     

Prevalence and diversity of Lyme borreliosis bacteria in marine birds

A potential role of seabirds in spreading Lyme disease (LB) spirochetes over large spatial scales was suggested more than 10 years ago when Borrelia garinii was observed in marine birds of both hemispheres. Since then, there have been few studies examining the diversity of Borrelia spp. circulating in seabirds, or the potential interaction between terrestrial and marine disease cycles. To explore these aspects, we tested 402 Ixodes uriae ticks collected from five colonial seabird species by amplification of the flaB gene. Both the average prevalence (26.0%+/-3.9) and diversity of LB spirochetes was high. Phylogenetic analyses grouped marine isolates in two main clades: one associated with B. garinii and another with B. lusitaniae, a genospecies typically associated with lizards. One sequence also clustered most closely with B. burgdorferi sensu stricto. Prevalence in ticks varied both among seabird species within colonies and among colonies. However, there was no clear association between different Borrelia isolates and a given seabird host species. Our findings indicate that LB spirochetes circulating in the marine system are more diverse than previously described and support the hypothesis that seabirds may be an important component in the global epidemiology and evolution of Lyme disease. Future work should help determine the extent to which isolates are shared between marine and terrestrial systems.     

吉林省长白及通化县莱姆病流行病学调查

目的了解吉林省长白及通化县林区人群、生物媒介蜱和宿主动物鼠的莱姆病螺旋体感染情况。方法在长白及通化县林区采集人群血清,并随机选择采样点进行捕蜱、捕鼠。采用间接免疫荧光法（IFA）检测血清,巢式PCR对蜱、鼠标本进行病原学检测,并通过基因测序方法确定基因型。同时蜱、鼠标本接种BSK培养基进行病原分离培养。结果从长白及通化县检查到人群血清莱姆病抗体IgG阳性率,长白县为12.04%（36/299）,通化县为9.35%（23/246）;蜱的带菌率,长白县为27.08%（39/144）,通化县为20.41%（20/98）;鼠的带菌率,长白及通化县均为10.00%（4/40,1/10）。通过序列同源性分析确定蜱和鼠携带的莱姆病螺旋体基因型存在Borrelia garinii。结论从血清学上证实长白及通化林区人群存在莱姆病螺旋体的感染,从病原学上证实全沟硬蜱和黑线姬鼠是当地莱姆病螺旋体的重要传播媒介和宿主。     

大兴安岭南段莱姆病的调查

1991年4～6月,我们在大兴安岭南段林区,对莱姆病进行了调查,应用间接免疫荧光抗体法检测496人居民血清,38人血清中抗莱姆病螺旋体IgG抗体效价≥1:128。根据临床表现和血清学调查,确诊莱姆病病例22例;从26只全沟硬蜱分离出4株莱姆病螺旋体。     

幽门螺杆菌flaB基因克隆及序列分析

目的：将幽门螺杆菌（Helicobacter，pylort，Hp）鞭毛蛋白B亚单位基因（月2B）克隆到质粒载体pNEB193上，并进行序列分析，为Hp疫苗的构建奠定基础。方法：用PCR方法扩增Hp月2B基因，并将其定向克隆入质粒pNEB193上，然后进行序列分析。结果：PCR扩增获得长度为1545bp的月2B基因片段，并成功克隆入pNEB193，序列分析结果显示扩增的月2B基因序列与Genebank公布的月2B基因序列基本一致。结论：本研究获得了月2B阳性克隆子，为Hp重组疫苗的研究奠定了基础。     

中俄,中哈边境地区莱姆病自然疫源地调查研究

目的：调查边境地区莱杂姆病和自然疫源地的状况。方法;对血清学、病原学、宿主、媒介,病例和地理景观方面进行调查。结果：用ＩＦＡ和ＥＬＩＳＡ法共检测人畜血清１５０１份,从１９９份人血清中检出抗体阳性１８份,阳性率为９．０５％,并发现莱姆病典型患者１５例;检查马血清２０份,阳性５份,阳性率为２５．００％;检查牛血清３９９份,阳性６９份,阳性率１７．２９％;检查羊血清８８３份,阳性１９９份,阳性率２２．５     

Occurrence of Borrelia burgdorferi in Ixodes ricinus in The Netherlands

In a 1989 surveillance to study the infection rate of Ixodes ricinus ticks with Borrelia burgdorferi, a total of 1838 nymphal and adult Ixodes ricinus ticks were collected from 20 locations in The Netherlands. By means of a culture method using modified Barbour-Kelly-Stoenner medium nymphs were examined in pools of 4 and adults were examined individually. With 63 out of 75 isolates propagation and serological identification were possible. All of these 63 isolates were identified as Borrelia burgdorferi by means of an indirect immunofluorescence assay. In nymphs the minimal infection rate was 2.4%. The rate of infection for adults was shown to be 14.3%. These results are in agreement with the percentages found by other investigators in West-Europe. In all locations examined Ixodes ricinus were found to be infected with Borrelia burgdorferi, although the infection rate varied. The results indicate that the infection is widespread among ticks in The Netherlands and the potential risk of acquiring Lyme borreliosis after a tick bite is serious.     

从四川白腹鼠体内分离出莱姆病螺旋体

1991年5～6月,我们在四川省南川县调查莱姆病动物宿主,从白腹鼠肾脏分离出1株螺旋体(SR1)。用种和属单克隆抗体对SR1菌株进行间接免疫荧光抗体试验,该菌株与美国B_(31)菌株的单克隆反应基本相似,但外表蛋白抗原并不完全一致,表明该鼠可能是我田南方莱姆病螺旋体的重要动物宿主。从白腹鼠分离出莱姆病螺旋体,在国内外属首次报告。     

105例拟诊莱姆病患者抗伯氏疏螺旋体抗体检查结果分析

目的 分析拟诊莱姆病患者血清学检查结果,明确莱姆病临床病症及区域分布情况,为有效诊治莱姆病提供科学依据.方法 应用间接免疫荧光试验(IFA)和ELISA方法,对来自全国各地的临床拟诊莱姆病患者进行血清抗伯氏疏螺旋体IgM和IgG抗体检查,并结合临床疾患进行综合分析.结果 2007-2008年共检查临床拟诊莱姆病患者105例,血清呈阳性反应者16例,阳性率15.24%.其中神经系统患者44例,阳性4例;心血管系统患者5例,阳性1例;皮肤病变25例,阳性6例;发热患者10例,阳性1例;关节痛14例,阳性2例;精神障碍疾患7例,阳性2例.16例患者应用抗生素治疗后有效率达87.50%.16例莱姆病患者分布在9个省(直辖市、自治区),确诊病例以黑龙江、吉林、内蒙古地区较多.结论 我国人群确实存在莱姆病螺旋体散发感染,通过血清学检查能及早发现病例,提高诊疗效率.     

Serological Detection of Lyme Borreliosis Agents in Patients From Korea, 2005–2009

Objectives

Laboratory tests are now being used to identify seropositive cases in patients suspected of having a Lyme borreliosis (LB) infection. From 2005 to 2009, we analyzed the serological and epidemiological characteristics of 53 LB positive cases in Korea using immunoblot assay.

Methods

During the five-year study period, a total of 1897 serum samples from suspected LB cases were referred to us for further laboratory diagnosis. The bacterial strains Borrelia afzeli pKo, Borrelia garinii 935T and Borrelia burgdorferi B31 were used for indirect immunofluorescent antibody assay. Immunoblot assay was performed using the recomBlot Borrelia.

Results

Based on the information from the clinicians, the main symptoms of LB infection were rash and fever (66.0%), neurological symptoms (30.2%), and arthritis (5.7%). Of the 53 cases, 16 (30.2%) were infected abroad and the remaining 37 cases (69.8%) were suspected to have been infected in Korea. Immunoblot assays detected high levels of the antigens p41 (FlaB) of B. burgdorferi and OspC of B. garinii in infected samples.

Conclusions

The causative bacteria of LB were not isolated from humans yet but from vector ticks and rodents in Korea, and a few cases were reported with serological diagnosis. Our results suggest that LB is present in all areas of Korea and indicate that B. garinii and B. burgdorferi may be the predominant bacteria in patients with LB. However, further studies are needed to isolate and identify the causative bacteria for LB in patients.     

用聚合酶链方法对北京林区莱姆病疫源地及莱姆病病原体基因型的探索性研究

目的：进一步明确北京林区是否存在莱姆病的自然疫源地及其分布，方法基于莱姆病螺旋体外膜蛋白A基因建立半巢式聚合酶链式反应(polymerase chain reactio,PCR)方法，对从北京6个林区采集的蜱和鼠进行检测和基因分型，选择阳性标准本进行克隆和序列测定，与已知序进行同源性比较，间接免疫荧光法检测抗莱姆病螺旋体IgG抗体，从长角血蜱中分离莱姆病螺旋体。结果：从门头沟区东灵山采集的标本中检测到莱姆病螺旋体DNA片段，3只游离全沟硬蜱1只检测阳性，57只寄全沟硬蜱若蜱中1只检测阳性；119只野鼠中9只检测阳性，其中8只，B.garinii阳性，1只B.afzelii阳性。50份野鼠血清有5份莱姆病螺旋体IgG抗体阳性，采集采的160只长角血蜱（20只／组）。未分离到莱姆病螺旋体菌株。结论：北京门头沟区东灵山可能存在莱姆病的自然疫源地，包括两个基因型，全沟硬蜱可能是莱姆病的传播媒体，野鼠可能是贮存宿主。